Microbes and Microbial Products as Herbicides - American Chemical

Chapter 2

Maculosin A Host-Specific Phytotoxin from Alternaria alternata on Spotted Knapweed Gary Strobel , Andrea Stierle , Sang Ho Park , and John 1

1,2

1

Cardellina

2,3

Department of Plant Pathology, Montana State University, Bozeman, M T 59717 Department of Chemistry, Montana State University, Bozeman, M T 59717 1

Downloaded by TUFTS UNIV on June 4, 2018 | https://pubs.acs.org Publication Date: September 25, 1990 | doi: 10.1021/bk-1990-0439.ch002

2

A strain of Alternaria alternata on spotted knapweed produces a family of diketopiperazines including maculosin, which is an L-tyrosine-L-proline analog. Maculosin is a host-specific toxin of Centaurea maculosa L. (spotted knapweed), the only one currently known from a weed pathogen. Current studies are underway to isolate and characterize a cytosol (soluble) protein receptor for maculosin in spotted knapweed. Affinity column chromatography employing maculosin covalently bonded to Sepharose 6-B has been used successfully to isolate an active protein fraction. Another phytotoxin produced by A. alternata, tenuazonic acid, acts in a synergistic manner with maculosin. Phytotoxin production in A. alternata seems to depend on one or more host plant metabolites. Each weed s p e c i e s i s a host t o an a r r a y o f p l a n t p a t h o g e n i c f u n g i and b a c t e r i a . Many o f t h e s e organisms induce d i s e a s e symptoms by v i r t u e o f p h y t o t o x i n p r o d u c t i o n (I). By f o c u s i n g on t h e s e p l a n t pathogens and on t h e i r p h y t o t o x i n s we hope t o f i n d n o v e l , e n v i r o n m e n t a l l y c o m p a t i b l e , h o s t - s e l e c t i v e c h e m i c a l agents f o r weed control. In t h e p a s t s e v e r a l y e a r s a number o f i n v e s t i g a t i o n s on t h e c h e m i s t r y o f p h y t o t o x i n s from weed pathogens has uncovered novel structures. The b i o l o g i c a l a c t i v i t y o f t h e s e compounds v a r i e d from n o n - s e l e c t i v e t o narrow host range p h y t o t o x i c i t y ( 2 , 1, 4 , 5, 6 ) . An i d e a l p h y t o t o x i n from a weed pathogen would have t h e unique p r o p e r t y o f e x p r e s s i n g host s p e c i f i c i t y a t t h e s p e c i e s 3

Current address: Center of Marine Biotechnology, University of Maryland, 600 East Lombard Street, Baltimore, M D 21202

0O97^156/90A)439-OO53SO6.00A) © 1990 American Chemical Society

Hoagland; Microbes and Microbial Products as Herbicides ACS Symposium Series; American Chemical Society: Washington, DC, 1990.


54

MICROBES AND MICROBIAL PRODUCTS AS HERBICIDES

or c u l t i v a r l e v e l . To d a t e , however, p h y t o t o x i n s w i t h t h i s extreme s p e c i f i c i t y are known o n l y from c r o p p l a n t pathogens ( 1 , 7 ) . Agroecosystems w i t h a s t r i c t l y homogeneous g e n e t i c b a s e , t y p i c a l o f c r o p p l a n t s , may s e r v e as a huge r e s e r v o i r o f e s s e n t i a l l y i d e n t i c a l p l a n t m a t e r i a l . A pathogen a t t a c k i n g such a c r o p , under t h e p r o p e r e n v i r o n m e n t a l c o n d i t i o n s , has t h e p o t e n t i a l t o produce an e p i d e m i c . Such an o r g a n i s m can be e a s i l y o b s e r v e d , i s o l a t e d and examined f o r p h y t o t o x i n p r o d u c t i o n . Common weed s p e c i e s , however, u s u a l l y e x i s t i n a p o p u l a t i o n w i t h a b r o a d , mixed g e n e t i c b a s e , and a r e o f t e n i n t e r m i n g l e d w i t h o t h e r p l a n t s . T h i s p r e c l u d e s t h e development o f such an e p i d e m i c and reduces t h e p r o b a b i l i t y o f d i s c o v e r i n g a weed pathogen c a p a b l e o f p r o d u c i n g h o s t s p e c i f i c t o x i n s . A t t h e o u t s e t o f t h i s s t u d y we chose t o examine C e n t a u r e a maculosa L. ( s p o t t e d knapweed), a s p e c i e s o f Immense economic importance i n t h e n o r t h w e s t e r n U n i t e d S t a t e s . T h i s p l a n t t h r e a t e n s forage production of m i l l i o n s of acres of rangeland i n t h i s region (S). I t a l s o has invaded c h o i c e r e c r e a t i o n a l l a n d s o f Y e l l o w s t o n e and G l a c i e r N a t i o n a l P a r k s . A f t e r an i n t e n s i v e s e a r c h f o r d i s e a s e d knapweed p l a n t s t h r o u g h o u t Western Montana, a s e v e r e l y d i s e a s e d p l a n t was found i n t h e e n v i r o n s o f B u t t e , Montana ( 9 ) . A p p r o x i m a t e l y 30% o f t h e l e a v e s were b l i g h t e d With l a r g e b l a c k brown n e c r o t i c l e s i o n s , and a t l e a s t 90% o f t h e f l o w e r s showed decay and e x t e n s i v e b l a c k m y c e l i a l g r o w t h . Three d i f f e r e n t f u n g i were i s o l a t e d from t h e l e s i o n s and p l a c e d i n t o pure c u l t u r e by t h e s i n g l e spore i s o l a t i o n t e c h n i q u e ( 9 ) . S u b s e q u e n t l y , each was t e s t e d f o r i t s a b i l i t y t o r e p r o d u c e d i s e a s e symptoms when i n o c u l a t e d onto a s p o t t e d knapweed p l a n t (£). Only one o f t h e s e f u n g i proved t o be p a t h o g e n i c and s a t i s f i e d a l l o f t h e c r i t e r i a o f K o c h ' s p o s t u l a t e s ( 9 ) . I t was i d e n t i f i e d as A1 t e r n a r i a a l t e m a t a . A l t h o u g h v a r i o u s form s p e c i e s o f A. a l t e m a t a a t t a c k i n g c r o p p l a n t s produce h o s t s p e c i f i c t o x i n s , none has been i s o l a t e d and I d e n t i f i e d t h a t s e l e c t i v e l y a t t a c k s weed s p e c i e s ( 1 0 ) . Mass l i q u i d c u l t u r e s o f our i s o l a t e o f A. a l t e m a t a were a n a l y z e d t o d e t e r m i n e i t s a b i l i t y t o produce p h y t o t o x i n s s e l e c t i v e t o s p o t t e d knapweed. I s o l a t i o n and I d e n t i f i c a t i o n o f D i k e t o p i p e r a z i n e s The p h y t o t o x i c e t h y l a c e t a t e s o l u b l e e x t r a c t s o f t h e f u n g a l c u l t u r e were chromatographed on Bio-Beads S-X4 ( h e x a n e - C H C l EtOAc, 4 : 3 : 1 , v / v ) . The r e s u l t i n g f r a c t i o n s o f t h i s chromatography, and a l l subsequent c h r o m a t o g r a p h i e s , were t e s t e d by t h e s t a n d a r d l e a f p u n c t u r e b i o a s s a y u s i n g s p o t t e d knapweed l e a v e s (2). F r a c t i o n s f o u r , f i v e , and s i x were p h y t o t o x i c . Size e x c l u s i o n chromatography o f f r a c t i o n f i v e on Sephadex LH-20 (CH CL MeOtBuiPrOH, 1:1:1, v / v ) y i e l d e d m a c u l o s i n , 1. S i m i l a r t r e a t m e n t o f f r a c t i o n s i x gave a m i x t u r e , which was r e s o l v e d by c e n t r i f u g a l c o u n t e r c u r r e n t chromatography (CHCl MeOH-H 0, 2 5 : 3 4 : 2 0 , v / v , l o w e r phase m o b i l e ) t o compounds 2 and 3 . S i z e e x c l u s i o n chromatography o f f r a c t i o n f o u r on B i o - B e a d s S-X8 ( C H C l - c y c l o h e x a n e , 3 : 2 , v / v ) y i e l d e d d i k e t o p i p e r a z i n e 4. The d i c h l o r o m e t h a n e s o l u b l e e x t r a c t s were permeated t h r o u g h Sephadex LH-20 (CH,CL -MeOH, 1:1, v / v ) . C e n t r i f u g a l c o u n t e r c u r r e n t chromatography as d e s c r i b e d above y i e l d e d compounds 5, 6, and 7. T a b l e I summarizes the i s o l a t i o n scheme f o r d i k e t o p i p e r a z i n e s 1-7. 2

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T a b l e I . Summary o f I s o l a t i o n D a t a , D i k e t o p i p e r a z i n e s

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"Final step in p u r i f i c a t i o n R e t e n t i o n t i m e i n hours °% y i e l d = mass o f compound obtained/mass o f t o t a l o r g a n i c e x t r a c t χ 100 Column 2x135cm; e l u a n t C H C l - M e 0 t B u - i P r 0 H ( 1 : 1 : 1 , v / v ) ; f l o w r a t e 1.9 mL/min 'Column volume 285 mL; e l u a n t CHCl -Me0H-H 0 ( 2 5 : 3 4 : 2 0 , v / v ) ; f l o w r a t e 4 mL/min; l o w e r phase m o b i l e Column 2.5x140cm; e l u a n t C H C l - c y c l o h e x a n e ( 3 : 2 , v / v ) ; f l o w r a t e 1.5 mL/min d

2

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57

AfoOiloSM

Only compounds 1 and 2 were p h y t o t o x i c t o s p o t t e d knapweed; 1 was a t l e a s t an o r d e r o f magnitude more t o x i c than 2. The m o l e c u l a r f o r m u l a o f 1 was determined t o be C H N p 0 by h i g h r e s o l u t i o n mass s p e c t r o m e t r y . P h e n o l i c and amide f u n c t i o n a l groups were i n d i c a t e d by i n f r a r e d a b s o r p t i o n s a t 3590, 3380 and 1670 cm , respectively. D e t a i l e d a n a l y s e s o f t h e mass s p e c t r a l f r a g m e n t a t i o n and H-NMR d a t a r e s u l t e d i n t h e p r o p o s a l o f s t r u c t u r e 1. The a b s o l u t e s t e r e o c h e m i s t r y was e s t a b l i s h e d by t h e s y n t h e s i s o f c y c l o ( L - P r o - L - T y r ) from the t-BOC d e r i v a t i v e o f L - p r o l i n e and t h e methylester of L - t y r o s i n e f o l l o w i n g l i t e r a t u r e procedures ( 1 0 ) . T h i s p r o c e d u r e i s known t o proceed w i t h o u t r a c e m i z a t i o n . The s y n t h e t i c and n a t u r a l m a t e r i a l s were i d e n t i c a l by H-NMR, mass s p e c t r o m e t r y , o p t i c a l r o t a t i o n and p h y t o t o x i c i t y (â). H-NMR and mass s p e c t r o m e t r y r e v e a l e d t h a t 2 and 3 were stereoisomers of composition C..H.JU) . Infrared spectroscopy i n d i c a t e d an amide m o i e t y and H-W1R d e c o u p l i n g p r o v i d e d t h e g r o s s structure cyclo-Pro-Phe. Comparison o f the ^-NMR s p e c t r a and o p t i c a l r o t a t i o n with those of s y n t h e t i c m a t e r i a l s l e d to the i d e n t i f i c a t i o n o f 2 as c y c l o ( L - P r o - L - P h e ) and 3 as c y c l o ( L - P r o - D Phe) ( 9 ) . The r e m a i n i n g f o u r d i k e t o p i p e r a z i n e s , 4 - 7 , were i d e n t i f i e d i n t h e same manner: m o l e c u l a r f o r m u l a e by mass s p e c t r o m e t r y and s t r u c t u r a l d e t a i l s from IR, mass s p e c t r a l f r a g m e n t a t i o n s and H-NMR decoupling experiments. 14

J6

3

1


1

2

1

Maculosin-Structure-Activitv Once t h e seven d i k e t o p i p e r a z i n e s had been i s o l a t e d and i d e n t i f i e d , t h e y were examined f o r p h y t o t o x i c i t y . The compounds were f i r s t a p p l i e d t o knapweed l e a v e s and h y p o c o t y l s f o l l o w i n g the d e s c r i b e d p r o c e d u r e s ( 2 ) . The most a c t i v e compound was 1, c y c l o ( L - P r o - L T y r ) , which produced l e s i o n s a t 1 0 ' , 1 0 ' , and 10 M. A t 10" M a s l i g h t n e c r o t i c f l e c k appeared i n 72 h o u r s , but t h i s was not c o n s i d e r e d a c t i v e by our s t a n d a r d s . Compounds 2 and 3, c y c l o ( L P r o - L - P h e ) and c y c l o ( L - P r o - D - P h e ) , d i f f e r e d i n p h y t o t o x i c i t y : the L, L d i a s t e r e o m e r induced n e c r o t i c l e s i o n s on knapweed l e a v e s a t 10" M, but t h e L, D isomer was not a c t i v e , even a t 10" M. Compounds 4, 5, 6 and 7 were not a c t i v e toward knapweed a t any o f t h e t e s t c o n c e n t r a t i o n s . These t e s t s were r e p e a t e d w i t h h y p o c o t y l t i s s u e , and s i m i l a r r e s u l t s were o b t a i n e d , a l t h o u g h i n 47% o f t h e s e t e s t s , c y c l o ( L - P r o - L - T y r ) induced l e s i o n s a t 1 0 ' ^ M . Comparison o f the d i k e t o p i p e r a z i n e s i s o l a t e d and t e s t e d i n t h i s s t u d y s u g g e s t s t h a t c e r t a i n f u n c t i o n a l groups a r e n e c e s s a r y f o r activity. M a c u l o s i n , t h e most a c t i v e s u b s t a n c e , p o s s e s s e s a p h e n o l i c m o i e t y , not uncommon i n p h y t o t o x i n s . The minor a c t i v i t y o f 2, c y c l o ( L - P r o - L - P h e ) , compared t o t h e i n a c t i v i t y o f i t s d i a s t e r e o m e r , s u g g e s t s t h e importance o f c o n f o r m a t i o n t o bioactivity. S t u d i e s on d i k e t o p i p e r a z i n e s c o n t a i n i n g p r o l i n e c l e a r l y showed t h a t L, L compounds assume an extended c o n f o r m a t i o n r e n d e r i n g both t h e a r o m a t i c m o i e t y and i t s h y d r o x y l group accessible for interaction with a biological s i t e ( H , 12). It is noteworthy t h a t the y i e l d s o f t h e v a r i o u s d i k e t o p i p e r a z i n e s were q u i t e c o n s i s t e n t i n r e p e a t e d f e r m e n t a t i o n s ; m a c u l o s i n was always t h e most abundant o f t h e d i p e p t i d e s . 3

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Maculosin-Bioactivitv A t t h i s p o i n t , m a c u l o s i n had been e s t a b l i s h e d as a p h y t o t o x i n o f s p o t t e d knapweed, but i t s h o s t s e l e c t i v i t y o r s p e c i f i c i t y needed t o be e s t a b l i s h e d . A t e s t d e s i g n e d t o e s t a b l i s h t h e h o s t - r a n g e o f t h e t o x i n u t i l i z e d t h e s i m p l e l e a f assay w i t h a wide v a r i e t y o f p l a n t s . Both monocots and d i c o t s were i n c l u d e d i n t h e t e s t r a n g e , i n c l u d i n g s e v e r a l c o m p o s i t e s . The a s s a y s were run a t ΙΟ" , 1 0 " , and 1 0 ' M. M a c u l o s i n c o n s i s t e n t l y induced n e c r o t i c l e s i o n s on knapweed l e a v e s , but i n no case was l e s i o n i n d u c t i o n observed on any o t h e r t e s t p l a n t . The n i n e t e e n t e s t p l a n t s were chosen a t random, a l t h o u g h an attempt was made t o i n c l u d e s e v e r a l c o m p o s i t e s and p l a n t s known t o be h o s t s t o o t h e r form s p e c i e s o f A l t e r n a r i a a l t e m a t a . Of p a r t i c u l a r i n t e r e s t i s the recent observation t h a t other species of C e n t a u r e a . i n c l u d i n g Ç. d i f f u s a . Ç. c v a n u s . and C. r e o e n s . were not a f f e c t e d by s y n t h e t i c m a c u l o s i n i n l e a f b i o a s s a y t e s t s a t 10" M. These h o s t - r a n g e t e s t s , w h i l e not e x h a u s t i v e , c l e a r l y e s t a b l i s h m a c u l o s i n as t h e most host s p e c i f i c weed p h y t o t o x i n on r e c o r d (see Table I I ) . Throughout t h i s s t u d y we used knapweed t e s t p l a n t s grown from seeds c o l l e c t e d from s e v e r a l g e o g r a p h i c l o c a t i o n s . Some d i v e r s i t y was seen i n t h e s u s c e p t i b i l i t y response o f p l a n t from d i f f e r e n t locales. Knapweed r e p r o d u c e s , i n t y p i c a l composite f a s h i o n , from wind-borne seeds c a r r i e d from t h e p a r e n t p l a n t . Seeds c o l l e c t e d from p l a n t s w i t h i n a twenty f o o t r a d i u s o f t h e o r i g i n a l d i s e a s e d specimen were most a f f e c t e d by m a c u l o s i n . Indeed, p l a n t s grown from seeds o f t h e s e p l a n t s a c t u a l l y e x h i b i t e d n e c r o t i c l e s i o n s a t ΙΟ" M i n l e a f b i o a s s a y t e s t s . Knapweed seeds were a l s o c o l l e c t e d from v a r i o u s l o c a t i o n s i n G a l l a t i n County and t h e B i t t e r r o o t V a l l e y , Montana. P l a n t s grown from t h e s e seeds were used i n t h e a s s a y s c i t e d i n T a b l e I I . One p l a n t was a c t u a l l y somewhat r e s i s t a n t t o m a c u l o s i n : n e c r o s i s was o n l y induced a t 10" M. These r e s u l t s were e x p e c t e d , s i n c e wide g e n e t i c d i v e r s i t i e s among p o p u l a t i o n s o f t h i s p l a n t most l i k e l y e x i s t . Such d i v e r s i t i e s would be e x p r e s s e d i n the s u s c e p t i b i l i t y range o f the p l a n t t o maculosin.


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M a c u l o s i n - Mode o f

Action

U n d e r s t a n d i n g t h e mode o f a c t i o n o f a h o s t - s p e c i f i c t o x i n i s o f interest. I t might p r o v i d e i n s i g h t i n t o t h e development o f p l a n t pathogen-host p l a n t dynamics (I). A l s o , the n a t u r e , l o c a t i o n , and f u n c t i o n o f t h e t o x i n r e c e p t o r - s i t e might be d i s c o v e r e d . This c o u l d e v e n t u a l l y l e a d t o a m o l e c u l a r g e n e t i c s approach t o weed c o n t r o l . A c r i t i c a l s t e p i n d o i n g such s t u d i e s i s t o have a v a i l a b l e i s o t o p i c a l l y l a b e l l e d t o x i n possessing a high s p e c i f i c radioactivity. Commonly, l a b e l l e d t o x i n i s p r e p a r e d v i a t h e a d m i n i s t r a t i o n of appropriate precursors to c u l t u r e s of the p a t h o g e n i c f u n g u s . U l t i m a t e l y , some s m a l l q u a n t i t y o f the l a b e l e d t o x i n i s o b t a i n e d which has a s p e c i f i c r a d i o a c t i v i t y one o r more o r d e r s o f magnitude l e s s than t h a t o f the p r e c u r s o r (low s p e c i f i c radioactivity). Methods f o r o b t a i n i n g t o x i n s o f an e x t r e m e l y h i g h s p e c i f i c r a d i o a c t i v i t y may i n v o l v e o r g a n i c s y n t h e s i s u s i n g


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STROBEL E T AL.

Maculosin

59

T a b l e I I . Summary, Host S p e c i f i c i t y S c r e e n i n g o f M a c u l o s i n Maculosin ( 1 ) : Concentration (M) 10"


b

DICOTS: C e n t a u r e a maculosa ( s p o t t e d knapweed) Lactuca s a t i v a (lettuce) C i t r u s 1imon (lemon) L v c o p e r s i c o n esculentum (tomato) Mai us s v l v e s t r i s (apple) H e l i a n t h u s annuus (sunflower) Cucumis s a t i v u s (cucumber) Euphorbia e s u l a ( l e a f y spurge) Bidens p i l o s a (marigold) Taraxacum o f f i c i n a l e (dandelion) Artemisia tridentata (sagebrush) C i r s i u m arvense (Canadian t h i s t l e ) E u p h o r b i a mi 1 i i Desmoul. (crown o f t h o r n s ) MONOCOTS: Sorghum halapense (johnsongrass) Poa annua (bluegrass) Avena s a t i v a (park o a t ) Agropvron repens (quackgrass) D i q i t a r i a ischaemum (crabgrass) Zea mays (corn)

Natural 10' ΙΟ"

3

4

+++

++

5

+

ΙΟ"

-

-

-

-

.

.

-

-

-

-

-

-

-

-

.

.

Synthetic 1 0 * 10" 5

+++

-

.

3

8

++

+

.

.

.

.

.

.

.

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

+++: weeping n e c r o t i c l e s i o n > 4 mm from i n o c u l a t i o n s i t e ; ++: n e c r o t i c l e s i o n 2-4 mm; +: n e c r o t i c l e s i o n 0.5-2 mm; -: no l e s i o n present or f l e c k < 0.5 mm. i n m o l a r i t y (M) a

b


60


p r e c u r s o r compounds p o s s e s s i n g h i g h s p e c i f i c r a d i o a c t i v i t y . Cm a c u l o s i n was s y n t h e s i z e d f o l l o w i n g the same p r o t o c o l as b e f o r e , u s i n g C - L - t y r o s i n e as a p r e c u r s o r (Ifi). C - m a c u l o s 1 n was p u r i f i e d by p r e p a r a t i v e TLC on s i l i c a g e l p l a t e s . The f i n a l p r o d u c t has i d e n t i c a l s p e c t r a l , c h r o m a t o g r a p h i c , and b i o l o g i c a l p r o p e r t i e s as t h a t o f the n a t u r a l p r o d u c t ( a ) . P r e p a r a t i o n s o f s y n t h e t i c m a c u l o s i n made i n t h i s manner have a s p e c i f i c r a d i o a c t i v i t y o f 0.025

Microbes and Microbial Products as Herbicides - American Chemical

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